The Theory of the Molecular Evolutionary Clock

Dr. Emile Zuckerkandl, 1986.

“It thus appears possible that there would be no evolution without molecular disease.”
-Linus Pauling. “Molecular Disease, Evolution and Genic Heterogeneity,” 1962.

In the early 1960s, Linus Pauling and Emile Zuckerkandl, a French postdoctoral fellow who had arrived at Caltech in 1959, began researching the characteristics of hemoglobin extracted from a number of different species of animals. Zuckerkandl used a technique called fingerprinting, a process taught to him by a Caltech graduate student named Richard T. Jones, to create patterns of the amino acid sequences in each hemoglobin molecule.

[In her Master of Science thesis (pdf link), Dr. Melinda Gormley described fingerprinting, which was invented by the English chemist Vernon Ingram, as “a two-step process, [that] utilizes paper electrophoresis and paper chromatography. It produces splotches on paper at various locations; each mark corresponds to a peptide (two or more linked amino acids).”]

Once patterns had been prepared for several species, Pauling and Zuckerkandl compared them two at a time, and it was from the results of these comparisons that the theory of the Molecular Evolutionary Clock was developed.

The Molecular Clock differs from other evolutionary theories in that it tracks the evolution of a molecule rather than the evolution of a species. The theory states that, every so often, a mutation occurs in a given hemoglobin molecule. Generally speaking, this mutation is the source of a molecular disease, but will not cause any significant change to any organism other than its host.

Occasionally, however, a mutation will cause a lasting alteration to the molecule, and as the organism with the altered molecule reproduces, the change becomes permanent. More alterations of this nature can then occur on top of the original modification, thus resulting in even more differences in those hemoglobin molecules that have descended from the mutated original.

It is these differences that Pauling and Zuckerkandl were interested in when they compared the fingerprint patterns of different species, and their research led to an important breakthrough. As the duo compared more and more fingerprint patterns in a wider range of combinations, they observed that the number of differences between fingerprints lessened as the two species became more closely related. Pauling later stated that

[Zuckerkandl] found that in the beta chain of the human and the beta chain of the horse, for example, 20 of the 146 amino acids are different; but with human and gorilla, only one is different. It is the same amount of difference, just one amino acid residue, as between ordinary humans and sickle cell anemia patients, who manufacture sickle-cell-anemia hemoglobin.

From there Pauling and Zuckerkandl proposed that the comparative-fingerprinting method could be used to speculate as to how long ago any two species deviated from a common ancestor. Even more specifically, they reached the conclusion that one amino acid would be substituted every eleven to eighteen million years for any given species.

The evolutionary theory of the Molecular Clock was not readily accepted by scientists because it proposed a constant rate of evolution. However, it’s importance has now been noted and more research has been done on Pauling and Zuckerkandl’s original work. See, for instance, the very thorough examination conducted by Dr. Gregory J. Morgan in his 1998 paper “Emile Zuckerkandl, Linus Pauling, and the Molecular Evolutionary Clock, 1959-1965.” [pdf link], as well as Naoyuki Takahata’s “Molecular Clock: An Anti-neo-Darwinian Legacy,” [Genetics, (May 2007) 176: 1-6; not freely available online] which concludes that “a molecular clock is a most remarkable manifestation and a tribute from nature to anyone who studies evolutionary biology.”

For more information on Pauling’s hemoglobin work, please visit the website It’s in the Blood!  A Documentary History of Linus Pauling, Hemoglobin and Sickle Cell Anemia, and for more on Linus Pauling, check out the Pauling Online portal.